Lin, H. H., Cao, D. S., Sethi, S., Zeng, Z., Chin, J. S., Chakraborty, T. S., Shepherd, A. K., Nguyen, C. A., Yew, J. Y., Su, C. Y. and Wang, J. W. (2016). Hormonal modulation of pheromone detection enhances male courtship success. Neuron 90: 1272-1285. PubMed ID: 27263969Summary:
During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. This study shows that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, this study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness.

Wu, Z., Guo, W., Xie, Y. and Zhou, S. (2016). Juvenile hormone activates the transcription of Cell-division-cycle 6 (Cdc6) for polyploidy-dependent insect vitellogenesis and oogenesis. J Biol Chem 291: 5418-5427. PubMed ID: 26728459Summary:
Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. The JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust. This study has investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. Cdc6 was shown to be expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. These data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.

He, Q., Zhang, Y., Zhang, X., Xu, D., Dong, W., Li, S. and Wu, R. (2016). Nucleoporin Nup358 facilitates nuclear import of Methoprene-tolerant (Met) in an importin beta- and Hsp83-dependent manner. Insect Biochem Mol Biol 81: 10-18. PubMed ID: 27979731Summary:
The bHLH-PAS transcription factor, Methoprene-tolerant (Met)1, functions as a juvenile hormone (JH) receptor and transduces JH signals by directly binding to E-box like motifs in the regulatory regions of JH response genes. Nuclear localization of Met is crucial for its transcriptional activity. It has been shown that the chaperone protein Hsp83 facilitates JH-induced Met nuclear import in Drosophila. However, the exact molecular mechanisms of Met nuclear transport are not fully elucidated. Using DNA affinity chromatography, binding of the nucleoporin Nup358, in the presence of JH, has been detected to the JH response region (JHRR) sequences isolated from the Kruppel-homolog 1 (Kr-h1) promoter. This study demonstrated that Nup358 regulates JH-Hsp83-induced Met nuclear localization. RNAi-mediated knockdown of Nup358 expression in Drosophila fat body perturbs Met nuclear transport during the 3 h after initiation of wandering, when the JH titer is high. The accompanying reduced expression of the transport receptor importin β in Nup358 RNAi flies could be one of the reasons accounting for Met mislocalization. Furthermore, a tetratricopeptide repeat (TPR) domain at the N-terminal end of Nup358 interacts with Hsp83 and is indispensable for Met nuclear localization. Overexpression of the TPR domain in Drosophila fat body prevents Met nuclear localization resulting in a decrease in JHRR-driven reporter activity and Kr-h1 expression. These data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin β and Hsp83.

Rahman, M. M., Franch-Marro, X., Maestro, J. L., Martin, D. and Casali, A. (2017). Local Juvenile Hormone activity regulates gut homeostasis and tumor growth in adult Drosophila. Sci Rep 7(1): 11677. PubMed ID: 28916802Summary:
Hormones play essential roles during development and maintaining homeostasis in adult organisms, regulating a plethora of biological processes. Generally, hormones are secreted by glands and perform a systemic action. This study shows that Juvenile Hormones (JHs), insect sesquiterpenoids synthesized by the corpora allata, are also synthesized by the adult Drosophila gut. This local, gut specific JH activity, is synthesized by and acts on the intestinal stem cell and enteroblast populations, regulating their survival and cellular growth through the JH receptors Gce/Met and the coactivator Tai. This local JH activity is important for damage response and is necessary for intestinal tumor growth driven by activating mutations in Wnt and EGFR/Ras pathways. Together, these results identify JHs as key hormonal regulators of gut homeostasis and open the possibility that analogous hormones may play a similar role in maintaining vertebrate adult intestinal stem cell population and sustaining tumor growth.

Shin, S. W., Jeon, J. H., Jeong, S. A., Kim, J. A., Park, D. S., Shin, Y. and Oh, H. W. (2018). A plant diterpene counteracts juvenile hormone-mediated gene regulation during Drosophila melanogaster larval development. PLoS One 13(7): e0200706. PubMed ID: 30011330Summary:
Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. This study demonstrates that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation.

Interestingly, the CAX females slowly became attractive so that by
96 h, time to onset of copulation was similar to controls. At this
time the C27 monoene and the 7,11-C29 diene were still significantly
reduced, whereas the C27 dienes were significantly increased, suggesting that there was a change in the CHC blend. The C27 dienes at 96 h may act alone or together with other
CHCs in the pheromone blend to increase female attractiveness
and decrease time to copulation in the CAX females (Bilen, 2013).

How does JH regulate CHC synthesis? CHCs are synthesized
from fatty acids via elongation, desaturation, reduction to aldehyde,
and oxidative decarbonylation reactions in oenocytes in
D. melanogaster. The developmental
appearance of the CHCs in CAX females in this
study clearly shows decreases in the long-chain n-alkanes (C23-
C27), the C25 and C27 monoenes, and the C27 and C29 dienes
with corresponding increases in the shorter-chain dienes. Similar
changes in diene profiles coupled with an increased time to
copulation was seen after reduction of Elongase-F in female
oenocytes (Chertemps, 2007). Reduction of Desaturase 1 in the oenocytes
caused the loss of both monoenes and dienes with a large increase
in the n-alkanes, whereas reduction of Desaturase-F
caused a loss of the female-specific dienes with a doubling of
monoenes and some increase in n-alkanes (Chertemps, 2006; Wicker-Thomas, 2009). Both manipulations
significantly increased time to mating. Thus, JH apparently
influences biosynthetic enzymes important in the synthesis of
the long-chain alkanes as well as Elongase-F and the desaturases.
These regulatory effects of JH on pheromone synthesis are similar
to its effects on aggregation pheromone synthesis in bark beetles.
In these beetles, JH III regulates the transcription of many of the
genes encoding the pheromone biosynthetic enzymes, especially
the genes encoding geranyldiphosphate synthase/myrcene synthase
(GPPS/MS), CYP9T2, and an oxidoreductase (Bilen, 2013).

Application of 0.64 pmoles of the JHM methoprene just after
eclosion rescued mating of the CAX females at 24 h after eclosion
to the level of about 31% as seen in the parental controls, and a 10-
fold higher dose caused about 58% to mate. After
treatment with the higher dose of methoprene, the 7- and 9-C27
monoenes significantly increased at 24 h, but the two
female-specific 7,11 dienes were not higher until 48 h. The
increased C27 monoenes which have been implicated as aphrodisiacs
(Marcillac, 2004) possibly made the CAX females more attractive at 24 h.
Whether there is also an effect of the exogenous JH on the maturation
of the female nervous system so that she becomes receptive
to male courtship earlier is not known and warrants further study.
At least three hormones (JH, ecdysone, and pheromone biosynthesis-
activating neuropeptide) have been shown to regulate
sex pheromone biosynthesis in different insects. This study has demonstrated that JH regulates sex pheromone 7,11-diene production in D. melanogaster females. Interestingly,
in another dipteran, the house fly Musca domestica, the primary
female sex pheromone is Z-9-tricosene.
Females ovariectomized immediately after eclosion do not synthesize
this compound, but synthesis is restored by either ovarian
implants or multiple injections of 20-hydroxyecdysone. These
two families of flies are evolutionarily distant, but the basis for
this difference in hormonal regulation is unknown (Bilen, 2013).

The duplication of the JH receptor Gce occurred in the higher
Diptera, and the two have partially redundant functions in
the larval fat body of D. melanogaster and at metamorphosis (Abdou, 2011).
Met plays a distinct role in adult optic lobe development during
metamorphosis, whereas Gce has no effect. Met is also
the predominant receptor required for the effects of JH on
ovarian maturation, both the normal timing and normal fecundity. This behavioral analysis of female mating and attractiveness
of Met and gce mutants indicates that JH is also acting
primarily via Met in its regulation of mating and pheromone
production. Surprisingly, in courtship assays using CS males,
Met27 females lacking Met were more attractive than the wildtype
CS females, whereas the CAX females were less attractive,
likely due to increased C27 dienes in Met27 females and decreased
7,11-C27 and -C29 dienes in the CAX females. These
findings suggest that JH acts mainly via Met in mating and
pheromone synthesis similarly to the roles of Met and Gce in ovarian
maturation where lack of Met delays maturation and reduces fecundity,
whereas lack of Gce has relatively little effect (Bilen, 2013).

This study has demonstrated that JH acting through its receptor
Met plays important roles in the initiation of sex pheromone
production and the maturation of female mating behavior in
Drosophila. Further investigation into these two aspects of JH
action -- in the peripheral tissues involved in sex pheromone production
and in the neuronal circuitry underlying the mating behavior --
is necessary to elucidate the details of its critical roles in
modulation of the onset of mating. An understanding at the molecular
level of this coordination in this Drosophila model should
shed insights into how hormones regulate pheromone production
and reproductive behavior in the vertebrates (Bilen, 2013).

Genetic evidence for function of the bHLH-PAS Protein Gce/Met as a juvenile hormone receptor

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog Germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. This study shows that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology (Jindra, 2015).

Reciprocal biosynthetic regulation between JH and ecdysone pathways has been demonstrated in several species, though the mechanisms segregate along taxonomic lines. In the hemimetabolous Diploptera punctata, expression of the ecdysone receptor proteins EcR and RXR (USP) coincides with an elevation in JH titer in 5-day old adult females; suppressing these genes lowers ecdysteroid titer and elevates JH biosynthesis in 6-day old females. In holometabolous insects, 20E has an evolutionarily-conserved allatotropic role. JH biosynthesis is irreversibly stimulated in cultured Aedes aegypti corpus allatum (CA) following 20-hydroxyecdysone (20E) addition at a concentration of 10-6 M. Fourth instar Bombyx mori corpus cardiacum-corpus allatum complexes likewise increased JH biosynthesis when cultured with 20E in a narrow concentration range, and this JH synthesis profile was concomitant with the elevation and subsequent decline in the transcript abundance of two JH biosynthetic enzymes. In both A. aegypti and B. mori, 20E overrides an inhibitory factor from the brain that prevents JH synthesis at a developmentally inappropriate time (Baumann, 2017).

In Drosophila such a factor(s) may be a product of the CA-LP1/2 neurons that innervate the CA, and which according to the present study, show JHR expression in WL3 larvae. Further, JH receptor expression in CA/PG cells, as well as in CA-LP and PTTH neurons suggests a potential role in coordinating hormone biosynthesis. For instance, by responding to changes in hormone titer, JHRs may participate in a feedback mechanism through which one or both receptors monitor the rate of biosynthesis or release of JH, ecdysone, or both. Experiments in other insects have shown that JH can inhibit PTTH release to indirectly suppress ecdysone production, or act directly on the PG to suppress ecdysteroid synthesis. Met and Gce both express in the PTTH cells and PG during the third instar. The coexpression of JH and ecdysone receptor machinery in the endocrine cells and associated neurons supports these tissues as critical sites for the convergence of signaling pathways, and the sensitivity of wvMet 27;;phm/TM6b flies to PG manipulation suggests that Met may be the key player in integrating these signals (Baumann, 2017).

In Drosophila oogenesis, JH is necessary for entry into the vitellogenic phase. The vitellogenic checkpoint occurs in stage 9 oocytes where JH is required for both yolk protein synthesis by the follicle cells and yolk protein uptake from the hemolymph. It also suppresses 20E-induced apoptosis of the oocyte at stages 8-9 when the females are starved. This apoptosis is mediated by the ecdysone-induced E75A. Both Met and gce are expressed in the follicle cells surrounding the primary oocyte in stage 8-10 egg chambers, which corresponds with the vitellogenic checkpoint). Met and gce in stage 8 and 9 follicle cells may mediate the action of JH, 20E, or both to oppose apoptosis of egg chambers. The comparatively severe oogenesis phenotype exhibited by Met mutants argues for a scenario under which Met-JH suppresses ecdysone-driven apoptosis, possibly by downregulating the apoptosis inducer E75A, which is a JH and 20E responsive gene, and a demonstrated transcriptional target of JH-Gce in cell culture. If gce regulates E75A expression in check-point stage egg chambers, gce overexpression, rather than loss of function, might be expected to promote apoptosis in egg chambers (Baumann, 2017).

Virtually nothing is known about Met and gce function in the brain, but a handful of studies have addressed the more general issue of JH action on nervous system development. Widespread and dramatic neurological damage results from a lethal dose of the JH analog methoprene in OR-C wildtype flies, including fusion of the subesophageal ganglion with the thoracicoabdominal ganglion, an incomplete fusion of the optic lobe neuropils with the central brain, and disorganization and degeneration of the optic lobe, particularly in the chiasma. Aross malformation of the lobula has been described in Met null mutants, evident as finger-like projections that encroach laterally into the inner chiasm, attributed partially to premature EcR B1 expression. gce mutants do not display this phenotype. Accordingly, gce expression in the developing optic lobe of WL3 larvae is sparse, and the T5 cells that arborize along the margin of the lobula likewise show Met but not gce expression, suggesting a potential maintenance function for Met-JH in the morphological integrity of the developing lobula (Baumann, 2017).

Conformational changes of recombinant DrosophilaUSP exposed to several different farnesoid compounds including natural JHs have been reported. JH III and JH I at 100 µm elicit the conformational changes to a similar degree whereas JH II is the least effective. Furthermore, by using Drosophila white puparial bioassay it has been have demonstrated that the biochemical differences in the three JHs mentioned above parallel the respective biological activity. For example, in prevention of adult emergence the 50% effective doses (ED50s) for JH I-III are 153, 678 and 143 pmol·puparium-1, respectively. The ED50 of methoprene has been reported as 5 pmol·puparium, and Met mutant flies are more resistant to another JHA, S31183, than the parental fly stock, suggesting the involvement of the Met locus in this resistance (Riddiford, 1991). Based on these studies, the order of efficacy of these compounds in this bioassay seems to be methoprene JH III ~ JH I > JH II. In contrast, the order was JH III JH II > JH I > methoprene in the current transfection assay. Methoprene is the most effective in the former and the least effective in the latter. This is not surprising since methoprene is often highly active over naturally occurring JHs when applied topically. For example, the early trypsin gene of Aedes aegypti is upregulated by JH, and low doses of methoprene, but higher doses of its native JH III are required to restore its expression in the ligated abdomens. The higher efficacy of methoprene in these bioassays may be due to its higher resistance to enzymatic degradation and possible higher penetration through the cuticle than natural JHs (Miura, 2005).

Another point is that JH I has been shown to be more active than JH II in the white puparial assay whereas JH II is more active in the current transfection assay. The difference between these two studies is the concentrations of JH used. In the current assay JH I and JH II were similarly much less effective than JH III in the physiological range while these differences were somewhat obscured at higher doses, although JH II was still more effective than JH I. At present this discrepancy is not explained. Possibly, there might be more than one pathway of JH signaling underlying in the white puparial bioassay, one mediated by USP and another by MET (Miura, 2005).

In the reporter assays, it was noted that unliganded MET repressed the intrinsic activation function possessed by GAL4-DBD. Although GAL4-DBD is believed to lack transactivation domains, it showed moderate transactivation potential in Drosophila S2 cells in this study. The nuclear localization of MET was confirmed by the finding that the MET-EGFP fusion is concentrated in the nuclei of transfected S2 cells. In addition, GAL4-DBD has a nuclear localization sequence. Therefore, it is reasonable to consider that the GAL4-DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and that the MET moiety is responsible for the observed repression. In the case of vertebrate Ahr, a multimeric complex including hsp90 anchors the unliganded Ahr in the cytoplasm, thereby preventing its transactivation function. Upon ligand binding, Ahr translocates to the nucleus and forms a transcription factor complex with Arnt. In fact, the C-terminal portion of Ahr fused to GAL4-DBD has been shown to act as a constitutive activator of gene regulation. Contrary to this, MET exists in the nucleus even in the absence of ligand. Upon ligand binding, it becomes a transcriptional activator. This resembles the ligand-dependent activation that has been shown in the activation function of many nuclear hormone receptors, rather than the case of the vertebrate Ahr whose activation function is regulated by its subcellular localization (Miura, 2005).

Two questions arise here as to whether MET functions as a homo- or heterodimer, and as to what DNA sequences are responsible for the binding of this transcriptional regulator complex. These questions are related since DNA-binding specificities of bHLH-PAS proteins are determined by their dimerization properties. For example, the dioxin receptor complex Ahr/Arnt heterodimer binds to TNGCGTG. Ahr recognizes the 5'-half-site TNGC, while Arnt recognizes the 3'-half-site GTG. Arnt is also capable of forming a homodimer that recognizes a consensus palindromic E-box sequence, CACGTG. Drosophila Sim protein forms a heterodimer with Tango (a Drosophila Arnt-like protein) and binds to ACGTG core sequence. Thus, DNA binding specificities of bHLH-PAS dimers are dependent upon the dimer configuration while Arnt or Tango always recognize the GTG motif. In the present study, the GAL4-DBD fusion of MET was used in transfection assays. Under these conditions MET is likely to behave as a homodimer because of its overexpression and because of dimerization interfaces provided by the GAL4-DBD moiety. Therefore, the natural dimerization partner and binding sequence of MET are unknown at present. Since the bHLH domain of MET shows relatively high similarity to vertebrate Arnts, the use of the consensus sequence CACGTG may be a good starting point to answer these questions (Miura, 2005).

Based on the framework by Wilson and coworkers, these results have further supported the notion that MET may function as a JH-dependent transcription factor. In further studies, identification of its target genes will help elucidate its in vivo function. Molecular dissection of MET and structural studies may lead to the development of new biologically active JHA and new strategies for pest management (Miura, 2005).

Metamorphosis of holometabolous insects, an elaborate change of form
between larval, pupal and adult stages, offers an ideal system to study the
regulation of morphogenetic processes by hormonal signals. Metamorphosis
involves growth and differentiation, tissue remodeling and death, all of which
are orchestrated by the morphogenesis-promoting ecdysteroids and the
antagonistically acting juvenile hormone (JH), whose presence precludes the
metamorphic changes. How target tissues interpret this combinatorial effect of
the two hormonal cues is poorly understood, mainly because JH does not prevent
larval-pupal transformation in the derived Drosophila model, and
because the JH receptor is unknown. The red flour beetle
Tribolium castaneum has been used to show that JH controls entry to metamorphosis
via its putative receptor Methoprene-tolerant (Met). This study demonstrates that
Met mediates JH effects on the expression of the ecdysteroid-response gene
Broad-Complex (BR-C). Using RNAi and a classical mutant, it has been
show that Tribolium BR-C is necessary for differentiation of pupal
characters. Furthermore, heterochronic combinations of retarded and
accelerated phenotypes caused by impaired BR-C function suggest that
besides specifying the pupal fate, BR-C operates as a temporal
coordinator of hormonally regulated morphogenetic events across epidermal
tissues. Similar results were also obtained when using the lacewing
Chrysopa perla (Neuroptera), a member of another holometabolous group
with a primitive type of metamorphosis. The tissue coordination role of BR-C
may therefore be a part of the Holometabola groundplan (Konopova, 2008).

In both Tribolium and Chrysopa, BR-C RNAi compromises the
larval-pupal transition without affecting earlier development, regardless of
the time of dsRNA injection. The TcBR-CKS342 homozygotes
die at the same stage. These data suggest that the moderate levels of
BR-C mRNAs, detectable during premetamorphic stages in both species,
has no essential role. This scenario would agree with the fact that zygotic
BR-C function is not required in Drosophila BR-C null
nonpupariating mutants until the onset of metamorphosis. However, as neither RNAi nor the likely hypomorphic TcBR-CKS342 allele present a complete loss-of-function situation, a possibility that BR-C plays some
additional role, not visualized by the phenotypes cannot be excluded. Importantly, the lethal phase correlates with a strong upregulation of BR-C expression. At
least in beetles, this stage coincides with a peak of ecdysteroid titer that
causes larvae to initiate prepupal development (Konopova, 2008).

In contrast to Drosophila npr1 mutants, metamorphosis was not
completely blocked by BR-C deficiency in Tribolium or
Chrysopa. Instead the arrested prepupae showed a blend of larval,
pupal, and partially even adult features. Based on the absence of the
pupal-specific gin traps in Tribolium and on the surface
microsculpture, the cuticle was apparently larval in both species, thus
confirming the requirement of BR-C for the pupal commitment of the
epidermis. Interestingly, although the thorny cuticle in Chrysopa BR-C(RNAi)
animals was distinctly larval, similar to in Tribolium, the body
pigmentation resembled that of pupae. It is not certain whether this mixed
character of the epidermis might be due to persisting CpBR-C function, or might be because CpBR-C is not necessary for the pupal pigmentation (Konopova, 2008).

Pupal characters in BR-C(RNAi) animals included rudimentary wings.
In particular, the weak phenotypes in Tribolium (produced with either
isoform-specific or diluted common-core dsRNAs) revealed that wing elongation
was highly sensitive to BR-C depletion. A similar effect of BR-C RNAi
was described for pupal appendages in Bombyx mori.
BR-C silencing prevented the gradual wing enlargement even in larvae
of the hemimetabolous milkweed bug Oncopeltus fasciatus.
Imaginal discs fail to elongate in Drosophila br mutants with
disrupted BR-C Z2 function. The short legs and wings are not due to insufficient
proliferation of the disc cells but are due to their inability to change shape
in response to the ecdysteroid. This cell shape change requires cytoskeletal
components whose mutations enhance the effect of br. The
rudimentary wings, present even in animals most severely affected by
TcBR-CKS342 mutation or by RNAi, suggest that cell shape
changes, rather than cell proliferation may be disrupted by the loss of BR-C
in Tribolium as well. Growing wings marked by EGFP in arrested beetle
prepupae support this idea. The legs in Tribolium BR-C(RNAi) animals were short also but were distally specified as pupal with two tarsal claws. By contrast, the arrested Chrysopa prepupae retained pretarsi with the larval-specific
elongated arolium, thus suggesting a stronger requirement for BR-C
function in the Chrysopa leg (Konopova, 2008).

Except for small deviations, gross morphology of Tribolium genital
segments with the pupal genital papillae was pupal in BR-C(RNAi)
animals. In addition, the larval-pupal transformation of the visual system was initiated,
as larval stemmata were replaced with ommatidia of the compound eyes. However,
as in Drosophila, TcBR-C was important for compound eye
differentiation. These observations suggest that not all aspects of pupal
development are completely blocked by BR-C depletion (Konopova, 2008).

While the above described structures are retarded in their development in
BR-C(RNAi) animals, others appeared accelerated in their development
towards the adult state, although none could be unambiguously defined as
adult. For instance, the antennae in Tribolium or the compound eyes
in Chrysopa resembled their adult counterparts, but in fact were
intermediates between pupal and adult organs. These heterochronic phenotypes
suggest that BR-C may not only be a pupal specifier,
but rather a temporal coordinator of the extensive morphogenesis in diverse
tissues during metamorphosis (Konopova, 2008).
Drosophila organs require a temporally regulated balance between
both inductive and repressive BR-C functions, represented by the individual
isoforms. Two alternative explanations are seen for the heterochronically advanced
phenotypes. First, these structures may require BR-C to repress precocious adult morphogenesis in them, but the inductive BR-C function is dispensable for development beyond larval state. Consequently, loss of BR-C accelerates their development. Second, if both functions are required but the repressive one is more sensitive to reduced BR-C dose, then the inductive function will prevail under an incomplete BR-C knockdown. The first alternative alternative is favored, because progression beyond the pupal stage seems to depend on BR-C downregulation (Konopova, 2008).

Periods of JH absence are required first in larvae to initiate the pupal
program, and later in pupae to exit it. BR-C in both cases promotes the pupal
fate, and therefore JH must regulate BR-C differently in
larvae and in pupae. In lepidopteran, as well as in Tribolium larvae,
JH prevents BR-C expression until the onset of metamorphosis, and
presumably that is how JH prevents pupal differentiation. Conversely, removal
of the JH source (allatectomy) causes both BR-C misexpression and
precocious pupal development. In pupae, ectopic JH induces BR-C, and in many
insects, including Tribolium, such JH application causes reiteration of the pupal stage. In Drosophila, BR-C misexpression alone is sufficient to inhibit adult cuticle formation. BR-C is therefore a prime target of JH signaling, but how JH
regulates BR-C expression is unknown (Konopova, 2008).

Precocious pupation, triggered by interference with the
putative JH receptor Met, coincided with precocious TcBR-C mRNA
increase in the sixth instar. Thus, disrupted JH signaling induced
TcBR-C similarly to allatectomy in lepidopteran larvae. As
expected, TcBR-C not only marked but also was necessary for the
untimely pupation, as TcMet; TcBR-C double-RNAi resulted in
a phenotype similar to TcBR-C RNAi alone, i.e. entry to a lethal
prepupal stage, except one or two instars too early.
Therefore, although the metamorphic program could be prematurely induced by
silencing of TcMet, it could not be completed without TcBR-C. However, loss of Met has been shown to worsen the effect of BR-C mutations in Drosophila, without altering BR-C expression. This again might reflect the different response to JH in the fly (Konopova, 2008).

The evidence that TcMet is required for regulation of
TcBR-C came from pupae, where the JH mimic methoprene induced
TcBR-C mRNA, but not after TcMet knockdown. This result
places TcBR-C downstream of TcMet in JH signaling.
Importantly, the averting of ectopic TcBR-C expression by
TcMet RNAi also rescued the methoprene-treated animals from repeating
the pupal stage and allowed them to become adult. Together, these findings suggest that, similar to in Drosophila, downregulation of BR-C is required to exit the pupal state in Tribolium (Konopova, 2008).

The following model for BR-C function in holometabolan metamorphosis. In larvae, JH acts through Met to prevent BR-C induction until the final
instar, when JH decline relieves the repression, and BR-C coordinates pupal
morphogenesis. Loss of BR-C function causes both retardation and acceleration
of development in diverse epidermal tissues, thus producing a mix of larval-,
pupal- and adult-like features. In early pupae, low JH titer normally allows
BR-C expression to drop, which is necessary for proper adult
differentiation. Exogenous JH, again acting via Met, causes BR-C misexpression, which in turn promotes another round of pupal, instead of adult, development. Whether Met regulates BR-C expression directly, and what determines whether BR-C will be repressed or activated requires further work (Konopova, 2008).

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) results in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), are significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, this study demonstrates that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis. However, as JH does not cause supernumerary larval molting in flies, evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented in this study, it is concluded that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult, abdominal cuticle is formed in Diptera. Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis. Similarly, global overexpression of jhamt (Juvenile hormone acid methyl transferase) results in severe defects during the pupal-adult transition and eventually death (Niwa, 2008). Third, CA ablation leading to JH deficiency causes precocious and enhanced fat body PCD. Fourth, JH deficiency results in pupal lethality and delayed larval development, although JH deficiency is not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. It is concluded that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the early-wandering (EW) stage. To address this question, it would be necessary to generate a mutant (i.e., of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway (Liu, 2009).

The insect fat body is analogous to vertebrate adipose tissue and liver and
functions as a major organ for nutrient storage and energy metabolism. In response to 20E pulses, Drosophila larval organs undergo a developmental
remodeling process during metamorphosis. Blocking
the 20E signal specifically in the fat body during the larval-pupal transition
(Lsp2>; UAS-EcRDN) prevented the fat body from
undergoing PCD and cell dissociation (Liu, 2009).

The experimental data in this paper demonstrates that JH prevents
caspase-dependent PCD in the fat body during the larval-pupal transition in
Drosophila. First, JH deficiency in Aug21>, UAS-grim
resulted in the fat body undergoing precocious and enhanced PCD and cell
dissociation. Aug21> is a GAL4 driver that specifically targets gene expression to the CA. Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals. Methoprene application on L3D1 was able to rescue ~40% of the pupae to adults, but it failed to rescue post-EW. Second, 2D-DIGE/MS
and qPCR analyses indicated that the fat body in the JH-deficient animals has
multiple developmental defects. The upregulation of the caspase genes
Dronc and Drice should account for the PCD in the fat body, as
overexpression of Dronc in the fat body causes PCD, cell
dissociation, and thus lethality. Overexpression of Dronc or Drice
in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the
20E-triggered transcriptional cascade in the fat body was downregulated in the
JH-deficient animals, indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body (Liu, 2009).

The antagonizing effect of JH on 20E-induced PCD in the fat body was
further confirmed in the JH-deficient animals by 20E treatment and RNA
interference of EcR. One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis. In the
JH-deficient animals, the upregulation of Dronc and Drice
resulted in precocious and enhanced PCD, such that the JH-deficient animals
are committed to die during the larval-pupal transition. This hypothesis was
strengthened by overexpression of Dronc specifically in the fat body,
which caused larval lethality. Taken together, it is concluded that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

Based on the phenotypes and gene expression profiles in the four fly lines
used, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD. First, the Met-overexpressing animals died during larval life, with precocious and enhanced PCD and cell dissociation in the fat body.
Dramatic upregulation of Dronc and Drice was observed when
Met was specifically overexpressed in the fat body and this
upregulation was significantly decreased by methoprene application demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing
animals exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD and the expression of
Dronc and Drice were upregulated but not as significantly as in the
Met-overexpressing animals. This might explain why the JH-deficient
animals did not die until early pupal life. Third, both the
global JH-overexpressing animals and the Met/gce-deficient animals
died during the pupal-adult transition. In these
animals, Dronc and Drice were downregulated and
caspase-dependent PCD was decreased in the fat body, implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc and Drice mutants die during
pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, it was also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (Liu, 2009).

In the future, it will be crucial to elucidate the detailed molecular
mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD.
In Drosophila S2 cells, the transcriptional activity of MET is
dependent on the JH concentration and both MET-MET and MET-GCE interactions can be greatly diminished by JH. The bHLH-PAS transcription factors typically function as hetero- or homodimers. If MET/GCE is the Juvenile Hormone Receptor (JHR), the transcriptional activities of
the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other
words, the dimerized MET/GCE should induce transcription of Dronc and
Drice and, in turn, JH binding to form the JH-MET/GCE complex should
reduce this induction. Although there are no examples in
the literature in which a receptor, without ligand, acts as a transcriptional
activator and the transcriptional activity of the receptor is diminished when
the ligand is bound, it could be speculated that the JHR is a unique hormone
receptor and perhaps that is the reason why it has yet to be isolated and
characterized. Unfortunately, the experiments described here were
conducted in Drosophila S2 cells, where the possibility of an
endogenous JHR could not be eliminated. Although MET/GCE is definitely a key
component in the JH signal transduction pathway, whether MET/GCE is the bona
fide JHR remains conjecture (Liu, 2009).

It is very likely that MET cross-talks with EcR-USP via a large molecular
complex. One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which is favored.
Drosophila FKBP39 (FK506-BP1) could be a key component in this
complex because it physically interacts with MET, EcR and USP, and binds the
D. melanogaster JH response element 1. Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy (Juhász., 2007).
Further analysis of the complex will be crucial to precisely define the
molecular mechanism of cross-talk between the action of JH and 20E (Liu, 2009).

In summary, it is concluded that JH counteracts MET and GCE to prevent
caspase-dependent PCD in controlling fat body remodeling and larval-pupal
metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires
further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium (Liu, 2009).

Juvenile hormone (JH) receptors, Met and Gce, transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified the chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with the PAS-B and bHLH domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of the larval fat body cells when the JH titer is low and JH induces Met nuclear import. Hsp83 was also accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuates JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to the reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR (He, 2014).

Drosophila S2 cells were screened for the presence of endogenous MET, but neither Western blotting with MET polyclonal antibody nor Met mRNA amplification by RT-PCR performed with S2 cell-derived RNA detected MET protein or mRNA. Therefore, the proteins of interest were introduced into S2 cells by expression of the respective cDNAs following transfection.
The interaction between MET and GCE proteins co-expressed in S2 cells was examined as GST-MET and GCE-V5 fusion proteins. Using a GSH pull-down technique, the GCE-V5 fusion protein was readily detected with V5 antibody as an immunoprecipitation product, demonstrating interaction between the two proteins (Godlewski, 2006).

Since MET is capable of binding JH in nanomolar concentration (Miura, 2005), the influence of JH treatment on MET-GCE interaction was examined. In the initial experiment, the transfected cell cultures were treated with JH III in final concentrations ranging from 10 μM to 1 nM for 1 h prior to homogenization and pull-down. JH III had no influence on the appearance of the MET-GCE protein complex. Conversely, analogous experiments with the JH agonists methoprene and pyriproxyfen revealed a disruptive effect of each of these juvenile hormone antagonists (JHAs) on the MET-GCE interaction. The strongest effect was observed at the highest concentrations examined. At the lowest concentration of 1 nM, pyriproxyfen showed no effect on the MET-GCE interaction, but methoprene showed a slight effect at 10 nM (Godlewski, 2006).

Both of the JH agonists are thought to be more resistant to catabolism by endogenous enzymes than is JH III. Therefore, the JH III treatment experiment was repeated using a series of time periods shorter than the previous 1-h incubation. At shorter incubation times, especially 15 min, JH III also disrupted MET-GCE interaction as did methoprene. However, the effect of JH III is reversible after 1 h, whereas methoprene requires 4 h for complete reversibility. At this shorter time period of 15 min for JH III, a dose–response effect is seen. The lowest detectable effect on MET-GCE interaction was 10 nM, an amount that is within the physiological concentration range for hormone action (Godlewski, 2006).

To determine if the hormone effect is specific to JH analogs, analogous experiments were carried out using farnesol and geraniol. Both of these compounds share overall chemical similarity with JH, but neither has JH activity in cell culture (Miura, 2005). Even when present in high concentration (10 μM) for the most effective time of JH III treatment (15 min), neither had an effect on MET-GCE interaction. Therefore, the effect on the interaction appears to be specific for JH III and JH agonists (Godlewski, 2006).

To determine if MET can form homodimers, S2 cells were co-transfected with a plasmid expressing GST-MET and one expressing MET-V5. Following incubation and V5 antibody immunoprecipitation, a strong band of MET was detected. To ensure that the MET-MET interaction was specific, a plasmid expressing MET was substituted for GST-MET and subjected to MET antibody detection following incubation and GSH pull-down. Since the MET antibody also recognizes the GST-MET fusion protein, two bands were seen on the Western blot, and the larger GST-MET band was far more intense, as expected. However, a band of the appropriate size for MET (79 kDa) was also evident. Therefore, MET-MET homodimers can form from different fusion proteins for MET and detected with different immunoprecipitation techniques (Godlewski, 2006).

When JH III was included for a 15-min preincubation, far less MET-MET was detected, similar to its effect on MET-GCE heterodimer formation. Therefore, MET homodimers can form in S2 cells, and they are also sensitive to the presence of JH III (Godlewski, 2006).

It was of interest to examine the effect of mutations of Met on the MET-GCE interaction. A variety of types of mutants were constructed, including severe truncations of Met, 10-amino acid deletions in conserved domains, and finally point mutations in each of the conserved domains. S2 cells were transfected with each mutant together with gce. Following incubation, GSH pull-down and V5 antibody detection of GCE were carried out to detect interaction (Godlewski, 2006).

Neither the truncated halves of MET nor the mutants with deletions in the bHLH and PAS-A domains interacted with GCE. The presence of JH III had no effect on the failure of interaction, as expected. However, Met mutants having a point mutation in either the bHLH (Met3) or PAS-A (Met1) domain expressed MET proteins capable of heterodimerization with GCE, and the presence of JH III greatly diminished the interaction. The Met128 mutant having an R433G point mutation in the PAS-B region showed a slight, almost undetectable interaction. So, it appears that severe mutations in Met as well as the R433G mutation in the PAS-B domain can block heterodimer formation or stability, but certain point mutations in the bHLH or PAS-A domains do not affect it (Godlewski, 2006).

Juvenile hormone (JH) plays crucial roles in many aspects of insect life. The Methoprene-tolerant (Met) gene product, a member of the bHLH-PAS family of transcriptional regulators, has been demonstrated to be a key component of the JH signaling pathway. However, the molecular function of Met in JH-induced signal transduction and gene regulation remains to be fully elucidated. This study, analyzing mosquito Met, shows that a transcriptional coactivator of the ecdysteroid receptor complex, FISC (Drosophila homolog: Taiman), acts as a functional partner of Met in mediating JH-induced gene expression. Mosquito Met and FISC appear to use their PAS domains to form a dimer only in the presence of JH or JH analogs. In newly emerged adult female mosquitoes, expression of some JH responsive genes is considerably dampened when Met or FISC is depleted by RNAi. Met and FISC are found to be associated with the promoter of the early trypsin gene (AaET) when transcription of this gene is activated by JH. A juvenile hormone response element (JHRE) has been identified in the AaET upstream regulatory region and is bound in vitro by the Met-FISC complex present in the nuclear protein extracts of previtellogenic adult female mosquitoes. In addition, the Drosophila homologs of Met and FISC (Taiman) can also use this mosquito JHRE to activate gene transcription in response to JH in a cell transfection assay. Together, the evidence indicates that Met and FISC form a functional complex on the JHRE in the presence of JH and directly activate transcription of JH target genes (Li, 2011).

Genetic studies have shown that Met is required for proper expression of JH target genes in fruit flies, red flour beetles, and mosquitoes. Although the protein structure of Met suggests that it may act as a JH-activated transcriptional regulator, the binding of Met to JH-responsive promoters has not been definitively demonstrated so far. In this study, a chromatin immunoprecipitation experiment indicated that Met was indeed associated with the early trypsin promoter when this gene was activated by endogenous juvenile hormone in the newly emerged adult female mosquitoes. This is a unique demonstration of Met directly regulating a JH target gene (Li, 2011).

To elucidate the molecular roles of Met in JH signaling, a number of proteins have been tested in vitro or in the cultured insect cells for their abilities to bind Met. The protein interactions with Met were largely independent of the presence of JH, or even repressed by JH. Using a library screening approach, a mosquito bHLH-PAS protein (FISC) was identified that binds to Met in a JH-dependent manner. EMSA and ChIP experiments have demonstrated that the Met-FISC complex forms in vivo and binds to a JH-regulated promoter in previtellogenic mosquitoes only in the presence of high titers of juvenile hormone. This observation is consistent with the RNAi results showing that both Met and FISC are required in adult mosquitoes for activation of JH target genes, such as AaET and AaKr-h1. The GBD-Met fusion (without the GAD-FISC fusion) activated the UASx4-188-cc-Luc reporter gene after the JH treatment. This activation also relied on the endogenous Taiman protein in the L57 cells; the JH induction was severely dampened when Taiman was depleted by RNAi. Formation of the Met-FISC complex thus constitutes a key step in signal transduction of juvenile hormone. It is also worth noting that not all of the JH target genes are affected by RNAi knockdown of Met or FISC, implying that JH might act through several distinct pathways even in a single tissue at a particular developmental stage (Li, 2011).

Transient transfection and gel shift assays indicated that Met-FISC activated the AaET promoter by binding to the JHRE. It is currently under investigation whether the two proteins are directly binding to the JHRE or are recruited to the JHRE via protein interaction with other transcription factors. Because of the relative large sizes of the two proteins, it is difficult to obtain full-length and functional recombinant Met and FISC proteins. EMSA experiments using in vitro-synthesized proteins turned out to be problematic, because both rabbit reticulocyte lysate and wheat germ extract displayed high background binding to the labeled JHRE. A preliminary study showed that the JH-induced transcriptional activation by Met-FISC was completely abolished in cell transfection assays if the DNA binding domain (bHLH region) of either Met or FISC was truncated. However, the possibility cannot be ruled out that the bHLH regions are also required for interactions with other proteins (Li, 2011).

A distal regulatory region of AaET was also shown to be indispensable for JH-dependent activation of the AaET promoter. Intriguingly, when four copies of JHRE were placed upstream of the minimal promoter (TATA box) of AaET, the JHRE seemed to be sufficient for the Met-FISC mediated JH activation. This discrepancy implies that regulation of JH target genes is more sophisticated than the binding of Met-FISC to JHRE. More studies are needed to elucidate the underlying molecular mechanisms (Li, 2011).

In vitro experiments have shown that Met can bind to both EcR and USP, two components of the ecdysteroid receptor. This study found that FISC, a coactivator of the EcR/USP, also binds to Met and plays an important role in juvenile hormone signaling. Whether these protein interactions are involved in the crosstalk of ecdysone and JH signaling is awaiting further experimental evidence. Because the binding of FISC to EcR/USP and Met relies on the presence of 20-hydroxyecdysone and juvenile hormone respectively, the shuffling of FISC between the two signaling pathways may account for the antagonistic actions of these two hormones (Li, 2011).

A sequence similar to the AaET JHRE is also found in the promoter region of AaJHA15, another JH-regulated gene in adult female mosquitoes. The common motif 2 discovered in a group of JH-activated Drosophila promoters also shares high sequence similarity with the AaET JHRE, suggesting an evolutionarily conserved mechanism underneath the JH-induced transcriptional activation. Indeed, the Drosophila Met and Taiman activated the 4×JHRE-luc reporter gene in a JH-dependent manner. Although DmMet-AaFISC appeared comparable to DmMet-DmTAI in mediating JH-induced gene expression, AaMet-DmTAI was completely unable to activate expression of the reporter gene after JH treatment. This observation suggests that the intricate protein interactions between Met and FISC/TAI determine the affinity of the dimers to the JHRE and/or their ability to activate transcription of the JH target genes (Li, 2011).

Unlike mosquitoes, two Met-like genes (Met and gce) exist in fruit flies. Combination of gce and Taiman also led to considerable activation of the reporter gene in response to JH. This observation is in line with a recent report showing that gce can partially substitute for Met in vivo (Baumann, 2010). It would be interesting to test next whether Met-TAI and gce-TAI preferentially bind to distinct JH responsive promoters in vivo (Li, 2011).

Mapping of the sequences directing localization of the Drosophila germ cell-expressed protein (GCE)

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e., the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. Confocal microscopy analysis was performed of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed characterization of the details of the subcellular traffic of this protein. GCE was shown to possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins Greb-Markiewicz, 2015).

Met is essential for the manifestation of the toxic and morphogenetic effects of JH/JHA in Drosophila (Wilson, 1986; Riddiford, 1991; Wilson, 1996; Restifo, 1998). Met mutants are resistant to these effects of methoprene (Wilson, 1986). MET can bind JH III with specificity and nanomolar affinity (Shemshedini, 1990a; Miura, 2005), suggesting that it is a component of a JH receptor. Met encodes a bHLH-PAS transcriptional regulator family member (Ashok, 1998) and MET can activate a reporter gene in transfected Drosophila S-2 cells (Miura, 2005; Wilson, 2005 and references therein).

Evidence was found for interaction between Met and BR-C as reflected by synergistically reduced viability and oogenesis seen in double mutants. Consistent results were seen with different combinations of Met and br or Met and rbp alleles, indicating that the interactions are not allele-specific in either direction. Met interacts with both the weak viable alleles br1 and rpb2 as well as the severe alleles br5 and rbp1 during pupal development. Each of the weak alleles possesses sufficiently functional gene product to permit completion of pupal development; but this amount is insufficient when MET is absent or defective. The more severe rbp1 homogygotes are pupal-lethal, but only at late metamorphosis, in the pharate adult stage. Lethality was shifted in rbp1Met27 pupae to prepupal/early pupal development, suggesting that MET absence causes the rbp1 product to be inadequate during these earlier stages in pupal development. Homozygotes of br5 and 2Bc die in the early and late prepupal stage, respectively, and the double mutants with Met27 show a similar phenotype, demonstrating that the interaction cannot shift lethality to an earlier stage, late third-instar larvae. These observations are consistent with the interaction between BR-C and Met beginning in prepupal or early pupal development. While the Met-BR-C interaction is interpreted as enhancing the lethality of br and rbp mutations, it is also possible that Met becomes an essential gene when BR-C function is reduced, or that the interaction is mutual, such that both mutations become more severe in phenotype when they are present together (Wilson, 2005).

Genetic interaction became strikingly evident when complementation failures between mutant alleles from different BR-C complementation groups occurred in the presence of Met27. Without MET, developing animals may be less able to make use of the partial functional redundancy among BRC isoforms. The interaction between mutant alleles of BR-C and Met is also evident in the adult stage when oogenesis is examined. Both the rate of oviposition and the paucity of vitellogenic oocytes in ovaries of br1Met27 and rpb2Met27 females reflects almost complete failure of oogenesis, with only a few eggs oviposited during the lifetime of the female (Wilson, 2005).

Previous studies have also detected BR-C interaction with other genes. Double mutants ofBR-C with another primary response gene, E74, show interaction for some but not all of the phenotypic characters. In addition to interactions among transcription regulators of the ecdysone cascade, br alleles interact with genes involved in imaginal disc morphogenesis, including those encoding an atypical serine protease, Stubble-stubboid, non-muscle myosin II heavy chain (Zipper), the Drosophila serum response factor transcription factor (Blistered), the small GTPase Rho1, cytoplasmic tropomyosin and 22 others. Although BR-C expression and function overlap the JH/JHA-sensitive period, methoprene treatment does not block
BRC expression in either wild-type or Met null mutants. Furthermore, the methoprene phenocopy, which excludes complementation group-specific defects (e.g., larval salivary gland persistence, which is rbp-restricted), is not consistent with methoprene simply reducing BRC expression. It is proposed that JH application results in abnormal function of BRC proteins, thus phenocopying certain characteristics common to all BR-C mutants. Therefore, the link between BR-C mutant phenotypes and JH-induced defects could be abnormal regulation of target genes, resulting in the phenotypic characteristics observed. Several possibilities have been suggested to explain methoprene pathology and BR-C phenocopy, including BRC interaction with an unidentified partner, perhaps MET (Restifo 1998). It is believed that the Met-BR-C genetic interaction can be explained best by this hypothesized protein-protein interaction between MET and BRC to regulate one or more target genes. Supporting this hypothesis are the following findings: first, both proteins are located in the nucleus, so there is no compartmental barrier to interaction. Second, both proteins appear to be transcription factors: BRC isoforms bind specific DNA sequences and regulate transcription. BR-C mutants have misexpressed secondary-response and other target genes. MET is a member of the bHLH-PAS family of transcription factors (Ashok, 1998) and was recently shown to act as one (Miura, 2005). Third, both are found at common times during development, such as prepupae and during vitellogenic oocyte development. Finally, PAS domains in bHLH-PAS proteins are thought to promote protein-protein interaction, either with other PAS proteins or as coactivators with nuclear receptor proteins, and the BTB/POZ domain of BRC has been implicated in proteinprotein interaction (Wilson, 2005 and reference therein).

In Met27 mutants, BRC protein accumulation profiles are normal. Since metamorphosis is not derailed in Met27 pupae, then BRC+ function in these pupae does not seem to be adversely affected. The fly may be protected from absence of MET by functional redundancy (Wilson, 1998). A candidate for the redundant substitute is the PAS gene germ cell expressed (gce), a gene with high (~70% amino acid identity) homology to Met that could substitute for MET to rescue larval and/or pupal development. However, this substitute does not appear to be satisfactory if BR-C is mutant. When a gce mutant becomes available, its phenotype could help evaluate this hypothesis (Wilson, 2005).

How does the application of exogenous JH act to phenocopy BR-C? It is clear that the action of these compounds occurs through MET, probably acting as a JH receptor component. JH is present during larval development when it presumably acts to prevent premature metamorphosis resulting from each wave of 20E secretion that triggers a molt. This fail-safe mechanism may occur by JH binding by and conformational change of MET, resulting in regulation of genes necessary for molting or perhaps simply blocking expression of metamorphic genes. Studies with Drosophila S-2 cells have implicated the transcription factor E75A in promoting JH regulation of larval development. At metamorphosis, when little or no JH is present, BR-C is expressed, and it is proposed that BRC dimerizes with the non-liganded MET protein to regulate a different set of target genes, promoting the initiation of metamorphosis. If exogenous JH is present during this time, it binds to MET and results in a more larval conformation, resulting in inappropriate binding to BRC and leading to a change in target gene expression patterns consequently seen as defects characteristic of BR-C mutants (Wilson, 2005).

Other work has implicated BR-C in the action of the JH agonist pyriproxyfen during metamorphic disruption. Application of this compound to white prepupae results in re-expression of BRC-Z1 in the abdomen during late pupal development, which in turn causes abnormal development of abdominal epidermis, including bristle disturbances. Those findings differ from those with methoprene in two significant ways. First, a lethal dose of methoprene causes a mild enhancement and prolongation of BRC protein accumulation in young pupae, but no re-expression at later times. Second, the modest effect of methoprene on BRC protein profiles cannot mediate the developmental effects of this JHA because the same mild persistence of BRC is seen in Met27 mutants, that are protected against methoprene-induced defects. It is not clear what underlies the difference in response of BR-C to methoprene and pyriproxyfen. It is noted that pyriproxyfen is a more powerful JH agonist than methoprene (Riddiford, 1991), but qualitative differences in the actions of the two compounds may exist as well (Wilson, 2005).

In summary, the results provide genetic evidence that supports other studies implicating BR-C as a focal point for interaction of JH and 20E signaling pathways, and they suggest that BRC and MET interact to regulate expression of one or more effector genes involved in metamorphic development (Wilson, 2005).

A role for juvenile hormone in the prepupal development of Drosophila melanogaster

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met27 larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis (Riddiford, 2010).

Insect molting and metamorphosis are governed primarily by ecdysone (used in the generic sense) and juvenile hormone (JH), with ecdysone causing molting and JH preventing metamorphosis. Juvenile hormone has a classic 'status quo' action in preventing the program-switching action of ecdysone during larval molts and in maintaining the developmental arrest of imaginal primordia during the intermolt periods. Its effects at the outset of metamorphosis, though, are more complex. Studies mainly on Lepidoptera show that for selected tissues JH needs to be present to allow them to undergo pupal differentiation, rather than undertaking a precocious adult differentiation (Riddiford, 2010).

The mechanism through which JH maintains the status quo and directs early development at metamorphosis is still poorly understood. Whether JH has one or multiple receptors, and the nature of these receptors, is still controversial. The best candidate for a receptor is the product of the Methoprene-tolerant (Met) gene, a PAS domain protein that was originally isolated in Drosophila melanogaster. In vitro transcribed and translated Met protein has been shown to bind JH with high affinity, and RNAi knock-down experiments in Tribolium castaneum show that Met is essential for mediating the status quo action of JH in this beetle (Riddiford, 2010).

In D. melanogaster, JH is thought to have no role in the onset of metamorphosis, since exogenous JH only delays but does not prevent pupariation. Although it has no apparent effect on the development of the imaginal discs, JH prevents normal adult development of the abdominal integument when given at pupariation. Internally, JH at this time affects normal reorganization of the central nervous system and development of the thoracic musculature. These effects of JH on metamorphosis do not occur in Met mutants, unless at least 100 times the dose is given. The Met27-null mutants proceed through larval development and metamorphosis apparently normally. However, if in addition, RNAi is used to suppress expression of Germ-Cell Expressed (Gce), a related bHLH protein with a high similarity to Met that heterodimerizes with it, Met-null mutants die as pharate adults. In the Met-deficient mutant, the adult eye shows a few (<12) defective ommatidia in the posterior region. Also, the females mature fewer eggs at a slower rate than do wild-type females, indicating that Met is also important for JH effects in egg maturation (Riddiford, 2010).

This study genetically allatectomized Drosophila larvae by targeting expression of a cell death gene to the corpora allata (CA), the gland that produces JH. These larvae form smaller puparia and showed precocious maturation of the visual system, but die around head eversion (Riddiford, 2010).

Although a number of studies have reported the effects of applying exogenous JH or JH mimics to Drosophila, there are only two very recent studies of the effects of manipulating endogenous JH on larval growth and metamorphosis, both of which appeared while this paper was under review. JH is normally present in the early larval instars, declines substantially during the last (third) larval stage and then returns transiently around the time of pupariation. The allatectomized (CAX) larvae undergo the expected two larval molts, but because sometimes the remains of degenerating CA cells are seen at the start of the last larval stage, nothing can be concluded about the requirements of JH for these larval molts. Recently, Jones (2010) using 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) RNAi to depress the level of JH and its farnesoid precursors in early larvae, showed that the larvae mainly die during the molt to the third instar, indicating that JH may be required for that molt (Riddiford, 2010).

The destruction of the CA by the third instar allowed examination of the role of JH during the last instar and early metamorphosis. The finding that these larvae were smaller than their CyO, UAS-grim siblings at pupariation could be explained by either the loss of JH or by the loss of the salivary glands, since these glands are also destroyed. Because dietary JH in the final instar rescued these larvae to normal size, the lack of the CA, rather than the lack of the salivary glands, is the cause of their reduced growth. Preliminary studies show that CAX larvae grow more slowly in the third instar, but the underlying basis for this retardation is not yet understood. Similarly, allatectomized third instar larvae display premature apoptosis of the fat body and downregulation of several enzymes involved in energy metabolism at the onset of wandering. These fat body effects could underlie the reduced larval growth seen in CAX larvae (Riddiford, 2010).

A major effect of the removal of JH was on the timing of events during the prepupal period. Studies on the wild silkmoth Hyalophora cecropia first showed that removal of the CA in the last larval stage resulted in the formation of a pupa with adult characteristics. Other moths, like Manduca sexta, showed more subtle responses to allatectomy, with premature adult differentiation most evident in the patterned region of the compound eye, posterior to the morphogenetic furrow. Subsequent studies on a variety of tissues in Manduca showed that the eye, the optic lobe and the ventral diaphragm each had a prolonged period of proliferation that extended from the prepupal period through early adult differentiation. This proliferation is maintained by α-ecdysone or low levels of 20-hydroxyecdysone (20E), but is terminated by high levels of 20E, which induces differentiation. These tissues are exposed to differentiation-inducing titers of 20E that occur during the larval-pupal transition early in their growth, but studies on the ventral diaphragm showed that JH 'protects' them from these high 20E levels, allowing them to continue proliferating. Removal of JH results in these tissues undergoing premature termination of tissue growth and precocious adult differentiation (Riddiford, 2010).

The response of Drosophila larvae to the loss of JH is in line with the effects seen in Manduca, and is also most evident in the developing visual system. In normal individuals, the appearance of EcR-B1 in the optic lobe and the termination of proliferation in the outer proliferation zone coincide with the ecdysteroid peak at head eversion and become more pronounced at 18 hours APF with the rise of ecdysteroid for adult differentiation. The separation of the R7 and R8 growth cones also begins about this latter time. The only one of these tissues that has been directly tested in vitro for 20E sensitivity is the optic lobe and in this case high levels of 20E do indeed suppress proliferation. It is assumed that the other processes also respond to the changing ecdysteroid levels. The lack of JH results in a heterochronic advance of these events by 10 to 12 hours, consistent with the tissues now responding to the earlier ecdysteroid peak that causes pupariation. Although the removal of JH advances these processes, it was found that the application of JH mimics delays them. Consequently, in selective tissues in Drosophila, JH acts to direct the nature of tissue responses to ecdysone (Riddiford, 2010).

The removal of JH or of one its receptors, Met, has a mixed effect on the developing visual system. No effect on proliferation or inductive events was seen in the eye disc itself, in that in CAX animals the morphogenetic furrow continues to move and similar rows of ommatidia have sent R8 axons into the medulla by 6 hours APF, as compared with controls. Likewise, in Met27 individuals there is only a slight advance (about 2 hours) in the schedule of lamina interneuron ingrowth into the medulla. However, for some of the cellular and molecular events, like the appearance of EcR-B1 and the separation of R7 from R8, there is a 10- to 18-hour advance in their occurrence. Hence, the lack of JH or of its receptor Met causes a heterochronic shift within the developing visual system with some differentiation responses being advanced relative to the normal schedule of neuronal birth and axon ingrowth. At least in the case of the photoreceptors, the effect of Met removal is largely cell autonomous, with the reduction of Met function in just those cells being sufficient to cause the precocious appearance of EcR-B1 and the early separation of R7 from R8. By contrast, the reduction of Met in lamina interneurons allowed these cells to precociously express EcR-B1 but did not affect the behavior of the R7 and R8 growth cones. This suggests that the separation of R7 and R8 is an active response of the photoreceptors, which is likely to be caused by the rising ecdysteroid titer driving adult differentiation. Although the lack of JH or Met function at the outset of metamorphosis results in the cell-autonomous expression of EcR-B1 in the photoreceptors, misexpression experiments show that the appearance of this receptor alone is not sufficient to bring about the early separation of R7 and R8. Therefore, although the upregulation of EcR-B1 is a prominent response to rising ecdysteroid titers, it is not the key change responsible for the repositioning of the receptor terminals (Riddiford, 2010).

As it is viable, the Met mutant allowed the final results of the mistiming of development in the optic lobes to be seen. No permanent effect was seen of the early separation of the R7 and R8 growth cones on the final anatomy of these projections in the medulla, or on the structure of the later neuropil. However, the lobula was grossly distorted and the normal layering of dendritic arbors disrupted. This aberrant morphogenesis also starts early, being already evident by 12 hours APF. The cellular basis for the lobula distortion, however, is not yet known (Riddiford, 2010).

Heterochronic shifts in the timing of development that extend beyond the visual system are likely to be the cause of the lethality seen in the CAX puparia. Puparia appear normal through the first 6 to 7 hours after pupariation but then abruptly undergo tissue collapse. In normal flies, the early part of metamorphosis is accomplished by a complicated replacement of histolyzing larval tissues by the growing adult tissues. Diverse tissues show individualized times of histolysis that are tied to the ecdysteroid titer. For instance, the larval midgut cells degenerate in response to the pupariation peak of ecdysone, whereas the larval salivary gland degeneration is triggered by the small rise of ecdysteroid at the end of the prepupal period. It is suspected that without JH, some of the histolysis events are mistimed, leading to the rapid death of the prepupa. It has been shown in CAX larvae that the fat body undergoes precocious programmed cell death beginning in the third larval instar. Interestingly, this lethal effect was not seen in animals in which Aug21-GAL4 drove RNAi for JH acid O-methyltransferase, the enzyme that converts JH acid to JH, in the CA (Niwa, 2008). Whether this indicates that JH acid plays a role in prepupal development or merely reflects the incomplete loss of JH in these animals is unknown (Riddiford, 2010).

All these effects of allatectomy can be rescued by JH either fed during the third instar or applied at the time of early wandering, but not at pupariation. A decline of JH III occurs in the third instar; this is followed by a peak of JH during late wandering. When JH begins to rise is unknown, as measurements were made every 24 hours. Presumably it is the lack of this JH during wandering when the ecdysteroid titer is rising and peaking that leads to the optic lobe anomalies and the premature histolysis (Riddiford, 2010).

The finding that the Met27 null mutant has the same defects in optic lobe development as are found in CAX prepupae strongly suggests that JH is acting via the Met pathway in controlling the timing of some events in the optic lobe. Accordingly, JHM treatment cannot suppress most of the premature development seen in prepupae lacking Met. However, a major difference between the CAX animals and the Met27 mutants is that the CAX prepupae died before head eversion, whereas the Met27 animals are viable. This difference is also seen in the precocious cell death of the fat body caused by allatectomy, which does not occur in the Met null mutant even in the presence of gce RNAi. Instead precocious cell death of the fat body was seen when Met was overexpressed in that tissue and the death could be suppressed by exogenous methoprene (a JH mimic). This latter finding suggests that JH would act in this case to suppress Met-mediated cell death. This idea was tested by seeing whether the removal of Met would protect the prepupa from the death caused by early allatectomy. When Met27; Aug21-GAL4>UAS-GFP/CyO females were crossed with UAS-grim males, 44% eclosed, all showing the CyO phenotype. The remainder died at head eversion, and should have been half CAX, Met-heterozygous females and half CAX, Met-null males. Another group was separated by sex prior to pupariation. Forty-nine percent of the females and 48% of the males died at head eversion. All of the adults that emerged were CyO, showing that all the CAX prepupae died regardless of whether or not they were lacking Met function (Riddiford, 2010).

These results together with the findings that JHM treatment of the Met27 mutant gave a partial rescue of the premature separation of R7 and R8, and of the decreased proliferation in the inner proliferation zone, indicate that there may be more than one receptor for JH. Thus, JH might act through multiple pathways. A major pathway involves Met, but Gce or some other mediator may serve as an alternate pathway in some tissues. A similar protective role of JH at pupation mediated by Met is found in Tribolium; injection of Met RNAi into either fourth instar larvae or final instar larvae caused the precocious appearance of adult eyes, adult antennae and other features in the resulting pupae (Riddiford, 2010).

These studies show that JH has an endogenous function in regulating Drosophila metamorphosis, a specific example being in orchestrating the timing of differentiation events in the developing visual system. These effects of JH are primarily mediated through the Met pathway. JH also is necessary for normal larval growth and has another, as yet undefined, crucial role in prepupal development that prevents death at head eversion. The latter effect is not mediated through Met, indicating that JH might act through multiple pathways (Riddiford, 2010).

Juvenile hormone (JH) is critical for multiple aspects of insect development and physiology. Although roles for the hormone have received considerable study, an understanding of the molecules necessary for JH action in insects has been frustratingly slow to evolve. Methoprene-tolerant (Met) in Drosophila melanogaster fulfills many of the requirements for a hormone receptor gene. A paralogous gene, germ-cell expressed (gce), possesses homology and is a candidate as a Met partner in JH action. Expression of gce was found to occur at multiple times and in multiple tissues during development, similar to that previously found for Met. To probe roles of this gene in JH action, in vivo gce over- and underexpression studies were carried out. Overexpression studies showed that gce can substitute in vivo for Met, alleviating preadult but not adult phenotypic characters. RNA interference-driven knockdown of gce expression in transgenic flies results in preadult lethality in the absence of MET. These results show that (1) unlike Met, gce is a vital gene and shows functional flexibility and (2) both gene products appear to promote JH action in preadult but not adult development (Baumann, 2011).

Is the rescue due to an abundance of GCE or to expression in tissue not normally expressing endogenous gce? To address the issue of the tissue specificity of Met/gce expression, recent results were used that demonstrated that larval fat body catabolism, required for completion of metamorphosis, is initiated by ecdysone, MET, and GCE and can be blocked by JH application. Perhaps high pupal survival of Met mutants following methoprene application results from an absence of methoprene-induced blockage of catabolism and the lowered pupal survival in Met27; gce transgenic flies results from substitution of GCE for the absent MET. Overexpression of GCE specifically in larvae fat body was carried out using a larval fat body GAL4 driver, and resistance to methoprene-induced mortality and male genitalia malrotation was examined. Methoprene-treated Met27; UAS-gce/lfb-GAL4 was found to be completely (50/50 examined) resistant to the male genitalia defect, indicating no blockage of the Met27 mutation. However, the progeny were only partially resistant to pupal death, showing that GCE can partially substitute for MET in this tissue to block the Met27 mutation and suggesting that tissue-specific, not widespread gce overexpression, may underlie the basis of the GCE substitution effect. Since the larval fat body shows little or no gce, then supplying GCE to this tissue can explain the tissue-specific effect seen in the pupal death phenotype. The eye phenotype was not rescued by larval fat body promoter-driven GCE, but it was completely rescued by compound-eye promoter-driven GCE. Therefore, GCE expressed in the larval fat body can partially substitute for MET in the tissue(s) responsible for pharate adult death, but not for eye or male genitalia, demonstrating tissue specificity of expression or utilization of GCE/MET (Baumann, 2011).

Little effect of gce overexpression was found on adult reproductive phenotypes of Met27. JH plays roles in both male and female reproduction in D. melanogaster as well as in many other insects. Clearly, overexpressed GCE can substitute for MET in Met preadults, but since the adult transgenic fly reproductive phenotypes were similar to those in adults not overexpressing the gce transgene, this substitution appears to be unproductive in adults. This result suggests that MET may be the major player in adults. The presence of only a single Met/gce homolog in three mosquito species and in the beetle T. castaneum with higher similarity to gce than to Met suggests that this reproductive role for MET evolved following the gene duplication seen in higher Diptera. Both Met and gce are present in the 12 species of D. melanogaster whose genomes have been sequenced; therefore, this duplication occurred earlier than the evolutionary divergence of these species that occurred as much as 60 million years ago (Baumann, 2011).

Clearly, gce underexpression can be lethal to either larvae or pupae, especially pupae, and especially in the absence of Met+. The presence of background Met+ allowed greater pupal development, shifting pupal death in RNAi individuals from the early pupal stage to the pharate adult/adult stage, depending on the promoter used and thus presumably on the level of RNAi produced. Underexpression of gce in the absence of Met+, in a Met27background, is more severe and results in a total loss of preadult viability. This suggests that both GCE and MET can interact to promote some vital aspect of larval/pupal development. Previous work using D. melanogaster S-2 cells has shown that MET and GCE can heterodimerize, suggesting a mechanistic basis for MET-GCE interaction. However, underexpression driven by the stronger tubulin promoter results in pupal death in a Met+ background, so the absence of Met+ is not a prerequisite for this phenotype. If both MET and GCE are necessary for JH action, then underexpression of both genes could result in death due to a failure of some JH-controlled developmental event, for example. The greater phenotypic severity of gce underexpression in a mutant Met background argues for the JH action scenario (Baumann, 2011).

Tissue-specific expression levels of Met and gce are given for 24 larval and adult tissues in the FlyAtlas database, determined by microarray analysis. The data show robust expression for gce in 12 tissues and for Met in 10 tissues. Seven tissues showed robust expression of both genes; interestingly, none are demonstrated JH target tissues. This could mean that (1) the presence of both GCE and MET are not required for JH action, (2) only one is required, or (3) few JH target tissues have been identified (perhaps the more likely explanation). However, there are some surprises in the FlyAtlas data set; for example, neither gene showed good expression in ovary or larval fat body, and only gce showed strong expression in male accessory glands, all demonstrated JH target tissues. Possibly, additional regulatory roles, independent of JH, for either of these transcription factors exist and are reflected in the FlyAtlas data set (Baumann, 2011).

Does this work show MET and GCE involvement in a JH receptor complex? Although the phenotypic characteristics of Met suggest involvement in JH reception, there is no direct evidence for involvement in a bona fide receptor. The disparate levels of transcript found for Met and gce in certain tissues might suggest separate roles for either or both of the gene products in certain tissues, and not formation of a mandatory heterodimer that might be predicted for a JH receptor. Indeed, one of the roles might involve eye development, which can be disrupted when either Met is mutated. Likewise, the lack of substantial resistance to methoprene in the Met+; UAS-gce dsRNA/actin-GAL4 flies might seem perplexing, considering the high resistance seen in Met mutants, but this conundrum might simply reflect a lack of strong JH binding by GCE, a possible requirement for resistance to the hormone and insecticide. Ligand binding might be the sole property of MET in a MET-GCE heterodimer, and loss of GCE does affect JH action, but not due to failure of JH binding. Future studies focusing on the role of gce may lead to more rapid progress in defining a JH receptor (Baumann, 2011).

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. This study demonstrates that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed (Bontonou, 2014).